Of 600,000 UK live births per annum, approximately 8,000 will be at birth weights below 1500 g, which equates to a gestational age of less than 32 weeks. Of these preterm babies, 1600 will die and a further 600 will develop cerebral palsy. The impact of disability increases dramatically when delivery occurs close to the limits of viability at around 24 weeks. The EPICURE study described how 25% of babies born before 25 weeks who survive to be discharged from hospital develop severe disability, 25% mild disability and less than 50% are developmentally normal at 30 months of age1. The economic impact of providing long-term health and social care for these families is therefore significant.
Preterm labour (PTL) is a syndrome, not a single disease process. Some aetiologies, for example placental abruption, are unpredictable and unpreventable. Maternal and/or fetal ‘stress’ may cause preterm labour by cortisol-mediated effects upon placental CRH. Multiple pregnancy causes preterm delivery both through placental CRH and through mechanical stretch of the uterus and cervix. But these are, in general, causes of ‘late’ preterm labour with less severe medical and economic sequelae.
In normal pregnancy, the onset of contractions is preceded by several weeks of cervical change characterised by decreased collagen and increased water content, identifiable clinically as effacement and shortening or cervical ‘ripening’. Cervical ripening is mediated by prostaglandin and cytokine secretion in the lower pole of the uterus and associated with an inflammatory cell infiltration. The later onset of uterine contractions is mediated by up regulation of a group of ‘contraction-associated proteins’ (CAPs) such as prostaglandin and oxytocin receptors, and gap junctions whose expression is repressed by progesterone. Preterm delivery prior to 32 weeks is associated with chorioamnionitis and ascending bacterial infection but recent studies have shown that most cases of early preterm labour cannot be attributed solely to ascending infection.
Classical cervical incompetence (secondary to a congenital weakness or acquired following destructive cervical surgery) is a cause of second trimester pregnancy loss and early preterm delivery, and it is now accepted that cervical competence is a continuum. In women whose cervix is short or weak, the biochemical processes of cervical ripening may occur because of stretch in the lower pole of the uterus. This leads to further softening and shortening of the cervix and so to a vicious cycle. Bacteria may then gain access to the uterus and therefore the final preterm delivery appears to be associated with infection although the initial initiating factors were not infection specific. There is a poor correlation between the inflammatory response, which stimulates preterm labour, and the number of bacteria present. In some women there is an exaggerated inflammatory response to trivial numbers of bacteria. This leads similarly to ripening and shortening of the cervix allowing further bacteria to gain access to the uterus and ultimately to a form of preterm delivery clinically indistinguishable to that associated with a weakened cervix. It is therefore possible that, if women at risk of early preterm labour can be identified, an intervention which effectively switches off the biochemical processes leading to cervical shortening may prevent or delay the onset of preterm labour and therefore significantly improve neonatal outcome.
Current approaches for the prediction of PTL are limited. PTL can be predicted in those known to be at increased risk by serial measurement of cervical length (CL) on trans-vaginal ultrasound. Women who have had a previous PTL, mid-trimester loss (MTL) or cervical cone biopsy are eligible for CL screening, though provision in the UK is not universal.
Women with cervical shortening are at increased risk of spontaneous preterm delivery, but remain asymptomatic until preterm labour is imminent. Early interventions to prolong pregnancy are available, but can only be delivered if obstetricians are aware that cervical shortening has occurred. Vaginal ultrasound can be used to diagnose cervical shortening, but is expensive and invasive, and therefore only available to a limited number of women attending specialist centres.
If the CL is found to be less than 25 mm there are two available interventions: cervical cerclage and progesterone treatment. Cerclage acts not only to support a weak cervix, but to retain the anti-bacterial mucous plug and prevent stretch mediated activation of inflammation. Progesterone treatment is effective and probably acts via inhibition of contraction associated proteins and nuclear factor kB expression.
However, CL surveillance clinics are labour intensive, expensive and do not provide care and intervention for women without pre-existing risk factors. In addition, using only past medical history to screen for eligibility lacks sensitivity and >80% of women attending such a service do not require any intervention and deliver at term gestations.
It has been suggested that routine measurement of cervical length at 18 to 22 weeks, linked to progesterone therapy, should be offered to the entire obstetric population12 13. However, this would be potentially costly.
A panel of biomarkers, routinely measured in all pregnancies, which are able to predict future cervical change or PTL itself would therefore be of great value in more accurately targeting pregnant women for surveillance and therapy. Only one biomarker is currently available to predict PTL; fetal fibronectin (fFN) in cervical or vaginal fluid. fFN is not useful in the distant prediction of early PTL because it is normally present in cervical secretions at up to 22 weeks gestation. Amniotic fluid or cervical secretion cytokines levels will also predict PTL but only close to the onset of labour2. All pregnant women currently undergo blood testing to screen for Rhesus group and viral infections at 13 weeks of pregnancy; this is an ideal time to screen for risk of early PTL and sufficiently early to allow enrolment in a surveillance program and delivery of an intervention.
Careful regulation of gene expression in the myometrium and fetal membranes is central to controlling the timing of labour onset. miRNAs are small, single-stranded, 19-25 nucleotide molecules that have emerged as important regulators of gene expression in almost all eukaryotes; a third of the protein encoding human genome is thought to be regulated by miRNAs. miRNAs are non-coding RNAs and function in a manner similar to small-interfering RNA to down-regulate gene expression at the post-transcriptional level. miRNA biogenesis involves a series of steps that lead to gene silencing. Briefly, miRNAs are transcribed in the nucleus as longer primary-miRNAs, which are cleaved to form hair-pin shaped precursor-miRNAs. These precursors are exported from the nucleus and further cleaved to form the mature miRNA which associates with the RNA induced silencing complex to target the 3′-untranslated region of specific mRNAs and inhibit their translation to protein. miRNAs are present in a cell free state in plasma and remain stable and easily measurable. Their potential utility as a biomarker of disease or response to treatment has consequently been widely acknowledged3.
miRNAs are expressed in a tissue specific manner and therefore their differential expression, both spatially and over time, is a potentially rich area of research. miRNA expression in the chorioamniotic membranes, placenta, umbilical cord and myometrium is currently being investigated by a number of groups. Cyclo-oxygenase 2 (COX2) (which catalyses the synthesis of prostaglandin which in turn modulates uterine contractions) is regulated at the post-transcriptional level through changes in specific miRNAs4. In addition, knockout studies of proteins essential for miRNA biogenesis have demonstrated that miRNAs play an essential role in reproduction. DICER is an RNAse III endonuclease that is essential for the biogenesis of miRNAs and small interfering (si)RNAs, and loss of DICER within ovarian granulosa cells, luteal tissue, oocyte, oviduct and potentially the uterus, renders murine females infertile5. In addition, disruption of the gene for Ago 2, another important component of RNA interference (RNAs) leads to embryo death early after implantation6. Intriguingly, placental miRNAs are also released into the maternal circulation. They remain stable and are easily detectable in blood and it has therefore been proposed that they might provide novel, non-invasive biomarkers for placental disorders such as preeclampsia or fetal growth restriction7.
Current understanding of how miRNA expression may regulate human myometrial gene expression and hence contractions, is limited. Renthal et al found that the miR-200 family of miRNAs is up-regulated in the labouring murine and human myometrium at term8. ZEB1 and ZEB2 were identified as two targets of this group in functional studies, and were found to be co-ordinately down-regulated in mouse models of PTL. ZEB1 and ZEB2 act as transcriptional repressors, which may inhibit the expression of contraction associated genes, oxytocin receptor and connexin-43, and block oxytocin induced contractility in cultured myometrial cells.
Williams et al linked miR-200a to progesterone metabolism through the repression of STAT5b, a transcriptional repressor of the P4 metabolising enzyme 20α-hydroxysteroid dehydrogenase, in the mouse and human uterus9. It is unclear how this is relevant to humans where labour is not associated with a reduction in circulating progesterone.
Recently, a study examining the global expression of miRNAs in cervical tissue from women following vaginal delivery at term or pre-labour LSCS, described 226 miRNAs expressed in the cervix10. Furthermore, miR-223, miR-34b and miR-34c were found to have increased expression with labour. Montenegro et al. examined miRNA expression in the fetal membranes in four distinct cohorts: term NL & L, and PTL with or without histological chorioamnionitis14. The authors detected 153 and 152 different miRNAs in at least 50% of samples in the term and preterm groups respectively. They found no difference in the term NL and term L groups, but described 13 miRNAs with reduced expression with advancing gestational age. They also found miR-223 and miR-338 had increased in expression in PTL membranes in the presence of inflammation.